We are studying the effect of ?-6 amino acid on the ? and ? assembly to produce tetrameric hemoglobin using time resolved spectroscopy. Hb S usually comprises about 40% of the total hemoglobin in the red cells of individuals with sickle cell trait. This lesser amount of Hb S compared to Hb A in persons with sickle cell trait, having a full complement of alpha genes is puzzling. In vitro ? and ? mixing experiments show that ?-? A dimers form more readily than the S dimer. The reasons for this are not readily apparent. The ?-6 amino acid is on the surface of the ? chain and far removed from the ?-1?-1 contact region that is critical for the initial formation of ??? dimers. We are now able to systematically test the effects of ?-6 amino acid charge and hydrophobicity on assembly of hemoglobin molecules by engineering various mutations at the ?-6 position. These studies will be important for understanding reasons for higher levels of the Hb S in sickle cell compared t o A type red cells. Furthermore, this difference is an important determinant of the clinical manifestations of sickle cell disease.